Comprehensive analysis of NRG1 common and rare variants in Hirschsprung patients

Abstract

Hirschsprung disease (HSCR, OMIM 142623) is a developmental disorder characterized by the absence of ganglion cells along variable lengths of the distal gastrointestinal tract, which results in tonic contraction of the aganglionic gut segment and functional intestinal obstruction. The RET proto-oncogene is the major gene for HSCR with differential contributions of its rare and common, coding and noncoding mutations to the multifactorial nature of this pathology. Many other genes have been described to be associated with the pathology, as NRG1 gene (8p12), encoding neuregulin 1, which is implicated in the development of the enteric nervous system (ENS), and seems to contribute by both common and rare variants. Here we present the results of a comprehensive analysis of the NRG1 gene in the context of the disease in a series of 207 Spanish HSCR patients, by both mutational screening of its coding sequence and evaluation of 3 common tag SNPs as low penetrance susceptibility factors, finding some potentially damaging variants which we have functionally characterized. All of them were found to be associated with a significant reduction of the normal NRG1 protein levels. The fact that those mutations analyzed alter NRG1 protein would suggest that they would be related with HSCR disease not only in Chinese but also in a Caucasian population, which reinforces the implication of NRG1 gene in this pathology.

Figure 1. Mutant NRG1 proteins.

Scheme of the main regions of NRG1 isoform ß1 protein and the location of the three variants analyzed.

Figure 2. Immunoblot analysis of NRG1 mutants.

Human NRG1 wildtype (WT) and mutants (M111T, M139I and R438H) were overexpressed in COS7 cell line. A) The intracellular levels of NRG1 wild-type and M111T, M139I and R438H mutants were detected with anti-neuregulin 1α/ß1/2 (F-20) antibody. All three mutants showed a significant lower protein expression. Alpha-tubulin was used as the loading control for normalization. B) The bar chart represents the quantitative data of the relative protein expression levels normalized with alpha-tubulin in three independent assays. (**p<0.005). C) The release of the extracellular domain in the medium was detected using anti-FLAG M2 antibody. The mutations did not affect the release of that domain of the NRG-1 protein. D) The bar chart represents the quantitative data of the relative protein expression levels normalized in three independent assays.

Figure 3. Analysis of cellular localization of NRG1 mutants.

Human NRG1 wildtype (WT) and mutants (M111T, M139I and R438H) were overexpressed in COS7 cell line. Cellular localization of NRG1 wild-type and mutants were analyzed using immunostaining with anti-neuregulin 1α/ß1/2 (F-20) antibody. Immunosignal for the three mutants was different in comparison with WT. Patchier and perinuclear distribution was observed in the three mutants in comparison with the punctuate at cytoplasmic membrane distribution of the WT protein.